Mammalian cells require a constant supply of oxygen to maintain energy balance. Hypoxia leads to reduced oxidative phosphorylation and depletion of cellular ATP, which can result in cell death. Sophisticated mechanisms have evolved which protect cells against hypoxia. A primary objective of our research is to identify the mechanisms that regulate hypoxia tolerance and survival in oxygen-sensing cells (such as those found in the carotid body). We have made extensive use of the oxygen-sensing PC12 cell line as a model system for this purpose. Preliminary investigations indicated that pre-exposure to mild hypoxia (i.e., "pre-conditioning") prevents cell death due to a subsequent exposure to severe hypoxia. We found that the Bcl-2 family of cytoplasmic proteins is involved in regulating this hypoxia tolerance in PC12 cells. The Bcl-2 family is composed of two opposing populations of proteins; those that promote apoptotic cell death, and those that oppose apoptosis. Our preliminary studies also indicated a role for the p38 kinase pathway in regulating hypoxia tolerance in the oxygen-sensing PC12 cell line system. Virtually nothing is known about the regulation of the Bcl-2 proteins during hypoxia or the genetic determinants that ultimately lead to hypoxia tolerance or cell death. In order to gain insights into this critical signaling process, we have developed a unique experimental approach that involves the use of our model cell line system and hypoxia-specific cDNA libraries to identify signal transduction pathways and genes that confer the hypoxia tolerant phenotype. These custom cDNA libraries contain more that 300 hypoxia-regulated genes that encode receptors, transporters, ion channels, metabolic and signal transduction enzymes, transcription factors, structural proteins, and proteins involved in regulating cell survival and death. The specific aims of the proposed study are: 1) Delineate the mechanism(s) by which the stress-activated p38 pathway is activated by hypoxia. A primary objective is to determine if the p38 kinase that is activated during hypoxia tolerance corresponds to p38alpha, p38beta, p38gamma, or p38delta. We will evaluate the effect of hypoxia on phosphorylation of the protein kinases immediately upstream of p38 (MKK-3 and MKK-6), and Rac, Ras, or Cdc42 are involved in the activation of p38 during development of tolerance to hypoxia. 2) Determine the role of the p38 kinase pathway in activation of pro-apoptotic and anti-apoptotic Bcl-2 family members. Our preliminary results indicate that both the p38 kinase pathway and the Bcl-2 family of pro-apoptotic and anti-apoptotic proteins are involved in conferring hypoxia tolerance to PC12 cells. We will examine interactions between the p38 kinase pathway and key members (Bcl-2, BcI-XL, 14-3-3, Bax, Bad, and Bnip-3) of the Bcl-2 superfamily during hypoxia. 3) Determine the role of p38 kinase and Bcl-2 family members in the regulation of hypoxia-responsive genes. We will use our hypoxia-specific cDNA libraries and custom microarrays to identify the genes that are regulated by hypoxia pre-conditioning and the role of the p38 kinase pathway and pro- and anti-apoptotic Bcl-2 proteins in the regulation of these genes. RNA interference will be used to evaluate the role of p38 and members of the Bcl-2 family in regulation of these genes. Results from this study will provide new insights into the mechanisms by which O2-sensing cells adapt and survive during hypoxia. This information may be helpful for developing strategies and targets for treating hypoxia-associated diseases and injury.